1. Field of the Invention
The invention is directed to a novel method of identifying toxicants in a sample. More particularly, the invention relates to a method of selectively determining the identity and the actual toxicity of toxicants in an aqueous matrix of water, wastewater or soil using antibodies.
2. State of the Art
The discharge of chemicals into the aquatic environment can result in measurable toxicity to aquatic organisms residing in the water column and/or aqueous sediments. These discharges are regulated by various state or federal agencies, which may require the discharger to perform testing to assure that the discharges are not causing toxicity to the aquatic environment. Accordingly, increased attention has been focused on methods of identifying the causes of toxicity in water subject to industrial and agricultural runoff to enable appropriate measures to be taken to control the source of the toxicant. These methods use a variety of approaches to separate toxic samples into different fractions and/or eliminate toxicity due to specific classes of chemicals.
In recent years, increased emphasis has been placed on the use of a standardized set of guidelines for determining the identity of toxicants in a water sample. The United States Environmental Protection Agency has produced a series of documents that address analysis of sources of chronic and acute toxicity, referred to as Toxicity Identification Evaluations ("TIE's") (see e.g., May 1992 EPA Publication, Technical Report 02-92; September 1993 EPA Publication, Technical Report 01-93; September 1993 EPA Publication, Technical Report 02-93). The TIE's utilize a three phase system, which is generally based on the concept of first testing an effluent in a bioassay for toxicity, subsequently separating out specific chemical constituents through either general chemical class separations or specific chemical separations, and retesting the sample to determine if the toxicity was eliminated due to the specific manipulation effected.
Phase I involves characterizing the general chemical classification of the chemical toxicant, e.g., oxidants, ammonia, volatile materials, polar or non-polar organic materials or cationic metals. The Phase I methods employ a series of chemical manipulations which are designed to remove or sequester materials belonging to one or more of the chemical classes described above or render them non-toxic. These manipulations include specific chemical and separative methodologies with a subsequent determination of whether a specific treatment has eliminated the toxicity of the sample. For example, EDTA and sodium thiosulfate additions, a graduated pH test, aeration and filtration manipulations, and the use of C-18 solid phase extraction (SPE) resin columns are generally used for Phase I categorizations.
Phase II is directed in determining the identity of the specific chemicals which have a role in the toxicity of the sample. In the September 1993 EPA Publication, Technical Report 01-93, the identification of toxicity due to organic chemicals is delineated in a multi-step process. First, the organics in the effluent sample are chemically separated according to their physical-chemical characteristics, generally by passing the effluent sample through a resin column which is sequentially eluted with an organic solvent or mixture of solvents into a series of fractions. Each fraction is then bioassayed and the fractions which continue to produce toxicity in the bioassay organisms are retained for further testing. Additional characterization of the toxic fractions is performed by combining and concentrating the toxic fractions on a resin column and subjecting this concentrate to additional fractionation using advanced analytical instrumentation, such as high performance liquid chromatography (HPLC). The individual fractions obtained by this process are bioassayed and those which produce toxicity are subjected to analysis by gas chromatography (GC), GC mass spectrophotometry (GC/MS) and/or HPLC/MS. If metals are indicated as being responsible for toxicity in the Phase I testing, e.g., with EDTA or similar chelator testing, then further analysis of these toxicants is necessary. The September 1993 EPA publication indicates that suitable testing procedures for the presence of cationic elements include the use of advanced instrumentation, such as atomic absorption analysis, inductively-coupled plasma-atomic emission spectroscopy, and inductively-coupled plasma-mass spectrophotometry.
The Phase III TIE process is essentially the same for both organic and metal toxicants. The overall goal of this phase is to determine if the candidate toxicant(s) identified in Phase I and 11 quantitatively account for the toxicity observed in the sample. The process consists of comparing the toxicity of the effluent sample with the candidate toxicants. In practice, this is accomplished by (1) measuring the level of toxicity in the sample, (2) measuring the level of each of the candidate toxicants in the sample, (3) measuring the toxicity of the candidate toxicants in "clean water", e.g., high purity water amended with appropriate salts to attain specifications for EPA Moderately Hard Water, or in a synthetic matrix similar to that of the sample and (4) summing the total toxicity of the candidate toxicants and comparing that with the total toxicity of the sample. If the values closely agree, the conclusion is that the candidate toxicants account for the toxicity of the sample.
These TIE testing methods have been found useful in many instances for determining the toxicity of a sample. In fact, when the Phase I and Phase II process is conducted in a technically competent manner, the results may often lead to the identity of one or more candidate toxicants. However, the methods utilized in the TIE Phase III process for the identification of specific organic non-polar toxicants and metal toxicants have several drawbacks.
One disadvantage of the present system is that there are many naturally-occurring substances in environmental samples which interact with toxicants to render a portion of the chemical toxicant biologically unavailable. Thus, comparing the concentrations of chemicals which cause toxicity in "clean water" with measured concentrations of the chemicals in environmental samples typically results in substantially more toxicity being predicted (based on measured chemical concentrations) than is actually measured by bioassay of the sample. This leads to erroneous results regarding the toxic components of the sample and, accordingly, the possibility of unnecessary or improperly calculated remedial measures taken to correct the problem.
Another disadvantage of present systems is that the analytical procedure used to identify candidate toxicants in a sample may not provide the required level of sensitivity to reliably detect the chemicals of interest in the sample matrix. For example, the level of accuracy and precision associated with procedures such as HPLC, GC and MS varies considerably due to the nature of the particular toxicant, the level of toxicant in the sample and the complexity of the sample matrix.
Another disadvantage of present systems is that specific toxicants may be lost during sample manipulation due to adsorption (i.e., onto resin columns), volatility and release of toxicant resulting in incomplete recovery of toxicant. Attempts to remedy such problems include estimating the amount of toxicant lost during the manipulations. However, these estimations are subject to error which can effect the results of the analysis.
Another disadvantage of present systems is their inefficiency in identifying additive, synergistic or antagonistic interaction effects caused by the presence of multiple toxicants in the sample. The inability of the prior art systems to identify such effects is due to the necessity of destroying the integrity of the sample itself in order to remove one or several toxicants. Using the prior art systems, it is not generally possible to obtain an accurate assessment of the actual toxicity contributed by a specific toxicant because the available methods do not permit selective removal of the specific toxicant without unintentional partial removal of other materials (other chemical toxicants and/or other components of the sample matrix). Accordingly, it is not possible to effect a direct comparison between the lethality caused by the toxicant alone at a concentration equivalent to that in the original sample or to determine if there is an interactive effect between multiple constituents of the sample.
Another disadvantage of present systems is that they may unintentionally and unknowingly add toxic materials to the sample matrix during the manipulations of Phase I and Phase II, resulting in false results.
Another disadvantage of present systems is that costly and intricate instrumentation is necessary to carry out the tests.
In contrast with the prior art systems described above, the present invention provides a novel method for the measurement of the toxicity of an environmental sample which avoids the above disadvantages. Importantly and surprisingly, Applicants method described herein overcomes the problems of the prior art by providing a highly sensitive and accurate system which properly assigns toxicity to various chemical components.